Oxygen free radicals mediate most ionizing radiation (“IR”) damage by attacking DNA bases and the sugar-phosphate backbone. This damage may result as adducts, other covalent modifications and strand breaks that scale directly with IR dose and intensity. Reactive oxygen species (ROS) induce at least twenty common adducts to purines and pyrimidines with 8-oxoguanine (8-oxodG) and 5-formyldeoxyuridine (5-FodU) (FIG. 1) among the most prominent products. Altered base pairing by 8-oxodG, 5-FodU and other lesions can induce mutations and promote cancers including those of breast, prostate, bone marrow, brain, lung, skin, colon, kidney, bladder, and others. One Gray unit “Gy” of absorbed radiation induces many thousands of base lesions along with double strand breaks in each cell, suggesting that dosimetry by direct measurement of base damage might be practical were a rapid, robust and accurate assay available. A promising approach is direct chemical detection of oxidative lesions. The reactivity of the aldehyde moiety of 5-FodU makes it a particularly attractive target because it is the only aromatic aldehyde to be identified in vivo and therefore is ideal as a biomarker.
Extensive literature is available examining the strengths and limitations of different methods of detecting oxidative DNA damage, as reviewed in Cadet et al., Mutat. Res. 531:5 2003; Dizdaroglu et al., Free Radic. Biol. Med. 32:1102, 2002. For this approach to be used in the development of a rapid field capable biodosimetry test, challenges include sufficient sensitivity, specificity, speed and reproducibility to reliably detect as few as 1 lesion in 105 bases from a readily available source of cells. In turn, the lability of some lesions as well as the potential for oxidative damage during work-up for the assay provides additional challenges. Given the wide range of different chemical species described, comprehensive analysis requires complete chemical digestion of purified DNA and analysis by mass spectrometry. For analysis of the adduct 8-oxoguanine (also 8-hydroxyguanine), it is common to use high pressure liquid chromatography (“HPLC”)-electrochemical detection or GC-MS to obtain quantitative results. Notably, the two methods can yield very different results from the same samples, which have been ascribed to the chemical derivitization required for the latter method. In turn, the requirement for purification of DNA, complete hydrolysis and complex analytic instrumentation suggest that these methods are not amenable to clinical use, let alone rapid dosimetry of mass casualties. Indirect methods include detection of glycosylase-sensitive sites by alkaline elution or comet assay. Both are compatible with analysis of cells rather than purified DNA, but neither offer sufficient sensitivity nor quantitation. Recent approaches such as detection anti-8-oxoguanine monoclonals (Bruskov et al., Biochemistery 64:803, 1999; Park et al., PNAS 89:3375, 1992), Fabs (Bespalov et al., Biochemistry 35:2067, 1996; Soultanakis et al., Free Radic. Biol. Med. 28:987, 2000) or binding of avidin (Struthers et al., Anal. Biochem. 255:20, 1998) similarly do not require DNA purification and are amenable to detection in cells and tissues (Persinger et al., Exp. Gerontol. 36:1483, 2001), but are subject to a range of artifacts and are relatively less sensitive to clustered damage. Commercial kits for 8-oxoguanine based on antibody, or avidin, detection by enzyme-linked immunosorbent assay (“ELISA”), imaging (Biotrin OxyDNA, Biotrin, Dublin, Ireland) and flow cytometry (HemoGenix OxyFLOW HemoGenix, Colorado Springs, Colo.) are available. However, these formats utilized with these assays, do not yield results over 2 Gy with accuracy and precision having a statistical certainty for rapid triage nor satisfactory for high throughput assays that provide accurate and precise results with a stated statistical certainty over the range of 0.5-10 Gy.
An attractive and comprehensive alternative is to use the chemical reactivity of some oxidized bases to detect their presence in purified DNA and/or in permeabilized cells. The Aldehyde Reactive Probe (“ARP”, N-(aminooxyacetyl)-N′-(D-biotinoyl) hydrazine (Ide et al., Biochemistry 32:8276, 1993; Kubo et al., Biochemistry 31:3703, 1992)) represents the most comprehensively studied chemical-based method to detect lesions on DNA caused by oxidative stress. ARP detects abasic sites on DNA by forming an oxime between a substituted aminoxy reagent and the aliphatic aldehyde produced on de-purination (FIG. 3). The reaction condenses a biotinylated aminoxy reagent to covalently link the biotin moiety to the oxidized DNA, and is amenable to quantitation (Kurisu et al., Nucleic Acid Res. Suppl. 45, 2001) and ELISA format (Kow and Dare, Methods 22:164, 2000). In addition to abasic sites, ARP also efficiently reacts with 5-FodU in DNA (Ide et al., 1993 supra) and it is possible that a significant fraction of the reactivity for ARP may derive from 5-FodU rather than abasic sites. ARP is commercially available from Dojindo (Rockville, Md.) and Invitrogen/Molecular Probes (San Diego, Calif.) and detection is performed by addition of either a streptavidin-HRP conjugate or a streptavidin-fluorophore conjugate. This method can be highly sensitive and has been used to detect individual lesions on single DNA molecules (Hirose et al., Photochem. Photobiol. 76:123, 2002; Kim et al., FEBS Lett. 555:611, 2003). However, it is a multi-step method and subject to background from other aldehydes such as carbonyl-modified proteins (Chavez et al., Anal. Chem. 78:6847, 2006). The binding between biotin and streptavidin has high sensitivity and specificity, but streptavidin is bulky and tetravalent, so that binding of a single streptavidin can mask several proximal biotins which can be expected at sites of cluster damage. Direct detection via Fluorescent Aldehyde Reactive Probe (“FARP”, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide (Chakrabarti et al., Int. J. Radiat. Biol. 75: 1055, 1999; Makrigiorgos et al., Int. J. Radiat. Biol. 74:99, 1998)) has been described, which removes the need for a binding step but still requires a washing step, ruling out homogeneous assays, and is subject to lower sensitivity and higher background. FARP probes are available from Invitrogen/Molecular Probes (San Diego, Calif.). A highly creative approach using a fluorescent resonance energy transfer (“FRET”) pair of FARP probes was shown to identify clustered damage (Chakrabarti et al., 1999, supra) and might be satisfactory to detect damage without extensive washing. Nonetheless, as yet, no methods let alone commercially available kits for using ARP or FARP probes with intact cells for cell imaging or flow cytometry are available.
Consequently, there is a need for a single sensitive, robust technology that meets the following criteria: (i) detection of a biomarker directly related to radiation exposure, (ii) low background levels from competing markers and reagents employed, (iii) able to detect exposure of at least 2 Gy in <30 minutes, (iv) ability to be adapted to multiple platforms such as lateral flow bioassay, flow cytometry and high throughput clinical analyzers (v) able to be adapted for field use in all environments without the need for refrigeration and minimal user training and (vi) acceptable cost.